MicroRNAs (miRNAs) regulate gene expression through an RNA interference (RNAi) mechanism by targeting specific messages and inhibiting their translation. The genes encoding miRNAs are longer than the processed miRNA molecule. miRNAs are first transcribed as primary transcripts or pri-miRNA and processed to short, approximately 70-nucleotide stem-loop structures known as pre-miRNA in the cell nucleus. This processing is performed in humans by a protein complex known as the Microprocessor complex, including the nuclease Drosha and the double-stranded RNA binding protein DGCR8. These pre-miRNAs are then processed to mature miRNAs in the cytoplasm by interaction with the endonuclease Dicer assisted by TRBP, which also initiates the formation of the RNA-induced silencing complex (RISC). This complex is responsible for the gene silencing observed due to miRNA expression and RNA interference. The pathway in plants varies slightly due to their lack of Drosha homologs. Instead, Dicer homologs alone affect several processing steps.
Efficient processing of pri-miRNA by Drosha requires the presence of extended single-stranded RNA on both 3′- and 5′-ends of a hairpin molecule. The Drosha complex cleaves RNA molecules at approximately two helical turns away from the terminal loop and approximately one turn away from basal segments. In most analyzed molecules this region contains unpaired nucleotides and the free energy of the duplex is relatively high compared to lower and upper stem regions. The resulting pre-miRNA has a short hairpin loop structure and is exported to the cytoplasm by Exportin 5 with help from cofactor Ran, a GTPase (Gwizdek et al., J. Biol. Chem. 278, 5505-8 (2003); Lund et al., Science 303, 95-8 (2004); Bohnsack et al., RNA 10, 185-91 (2004)).
When Dicer cleaves the pre-miRNA stem-loop in the cytoplasm, two complementary short RNA molecules are formed, but only one is integrated into the RISC complex on the basis of the stability of the 5′ end. The remaining strand, known as the passenger strand is degraded. After integration into the active RISC complex, miRNAs base pair with their complementary mRNA molecules and induce down regulation of the expression of the transcript by one of the two key mechanisms, depending on the degree of complementarity between the miRNA and the target mRNA. In animals, pairing between miRNA and their target mRNAs is not usually perfect, although there are a few exceptions where perfect or near perfect recognition exist (Yekta et al., Science 304, 594-6 (2004); Mansfield et al. Nat Genet 36, 1079-83 (2004)). If the complementarity between the miRNA and the target is perfect or near perfect, then the cleavage of the mRNA is mediated by the endonuclease (slicer) activity in the RISC provided by Ago2 protein. Where miRNAs bind to their targets via imperfect base pairing, miRNA bound messages may be directed to a cytoplasmic foci known as P-bodies or processing bodies where the ribosomes are depleted but rich in nucleases (Parker et al., Nature Structural & Molecular Biology 11, 121-12 (2004)). P-bodies serve as either degradation centers or storage depots for these messages, where their translation is inhibited.
To date, close to 500 miRNAs have been identified in humans (Griffiths-Jones, S. Nucleic Acids Res 32, D109-11 (2004)). Bioinformatics approaches have predicted that these miRNAs are capable of regulating at least 30% of human transcripts (Lewis, et al. Cell, 2005. 120(1): p. 15-20). As a result, miRNAs have the potential to play a vital role in many biological processes whose deregulation could lead to various disease states. Experimental evidence is accumulating to elucidate their roles in many biological processes. These attributes make miRNAs a potential class of targets for therapeutic intervention. However, the lack of current understanding on specific roles played by individual miRNAs in a plethora of biological processes has complicated the targeting of miRNAs.
Erythropoietin (EPO) is a glycoprotein hormone involved in the maturation of erythroid progenitor cells into erythrocytes. It is essential in regulating levels of red blood cells in circulation. Naturally occurring erythropoietin is produced by the liver during fetal life and by the kidney of adults. EPO circulates in the blood and stimulates the production of red blood cells in bone marrow. Anemia is almost invariably a consequence of renal failure due to decreased production of erythropoietin from the kidney. Recombinant erythropoietin produced by genetic engineering techniques involving the expression of a protein product from a host cell transformed with the gene encoding erythropoietin has been found to be effective when used in the treatment of anemia resulting from chronic renal failure.
Vascular endothelial growth factor (VEGF) is a positive regulator of angiogenesis. Hua et al., MiRNA-Directed Regulation of VEGF and Other Angiogenic Factors under Hypoxia, PloS ONE 1(1): e116, 1-13, 2 (2006). VEGF is a highly specific mitogen for vascular endothelial cells. Neufeld, Cohen et al., Vascular Endothelial Growth Factor (VEGF) and Its Receptors, FASEB J. 13, 9-22 (1999).
Low levels of erythropoietin are normally present in human urine, while individuals suffering from aplastic anemia exhibit elevated levels of urinary erythropoietin. The purification of human urinary erythropoietin by Miyake et al. in J. Biol. Chem., 252, 5558 (1977), used, as starting material, urine from aplastic anemic individuals. To date, however, urinary erythropoietin has not been shown to be therapeutically useful.
The identification, cloning, and expression of genes encoding erythropoietin are described in U.S. Pat. No. 4,703,008 to Lin. A description of a method for purification of recombinant erythropoietin from cell medium is included in U.S. Pat. No. 4,667,016 to Lai et al. The expression and recovery of biologically active recombinant erythropoietin from mammalian cell hosts containing the erythropoietin gene on recombinant plasmids has, for the first time, made available quantities of erythropoietin suitable for therapeutic applications. In addition, knowledge of the gene sequence and the availability of larger quantities of purified protein have led to a better understanding of the mode of action of this protein.
Given the known therapeutic benefits of EPO, methods of increasing EPO expression or secretion of EPO would be of great benefit to patients in need of EPO therapy. The methods and compositions described herein address these and other needs in the art.